Rheumatoid arthritis (RA) is characterized by the synovial inflammation of multiple joints. The affected synovial tissues contain activated macrophages, fibroblasts, T- and B-lymphocytes. The synovial fibroblasts proliferate and release tissue-degrading enzymes in response to pro-inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6, which are produced in situ (Arend, W. P. and J. M. Dayer, 1995, Arthritis Rheum. 38: 151-160; Feldmann, M. et al., 1996, Cell 85: 307-310). The resulting hyperplastic synovial membrane, termed pannus, irreversibly destroys the cartilage and bone of the affected joints.
Almost all of the conventional anti-rheumatic drugs and recently developed biological reagents are aimed primarily at the suppression of inflammatory mediators involved in RA. Most of the biological reagents are intended to neutralize pro-inflammatory cytokines, such as TNF-α, IL-1, or IL-6 (Elliott, M. J. et al., 1994, Lancet. 344:1105-1110; Moreland, L. W. et al., 1997, N. Engl. J. Med. 337: 141-147; Campion, G. V. et al., 1996, Arthritis Rheum. 39: 1092-1101; Arend, W. P. et al., 1998, Annu. Rev. Immunol. 16: 27-55; Yoshizaki, K. et al., 1998, Springer Semin. Immunopathol. 20: 247-259). Hydroxychloroquine, d-penicillamine, gold, sulfasalazine, immunosuppressive methotrexate, and such, are included as representative anti-rheumatic drugs. Although, target molecules responsible for their anti-rheumatic effects are not accurately known, they are considered to suppress the inflammatory processes (Conaghan, P. G. et al., 1997, Curr. Opinion Rheumatol. 9: 183-190). In contrast, newly developed drugs, such as leflunomide, cyclosporin A, and FK506 inhibit intracellular molecules essential for the activation of the immunocompetent cells involved in the rheumatoid synovitis. The TNF-α antagonists (such as remicade and infliximab) neutralize the inflammatory mediators released by the synovial cells. Most patients respond well to these drugs, but some are resistant or the effect of these drugs decreases after a period of disease remission in other patients. Moreover, there is little evidence proving that these drugs arrest the progression of the disease in the long run. It is presumed that the suppression of one inflammatory mediator might activate other mediators because of the complexity and redundancy of the inflammatory pathways. Therefore, it is believed that the total prevention of joint destruction is not available through conventional treatments. In contrast to this, the present inventors have investigated means for preventing the creation of the destructive synovium hyperplasia by directly inhibiting the growth of the synovial fibroblast itself using cell-cycle-suppressing proteins called cyclin-dependent kinase inhibitors (CDKIs).
The CDKI molecules consist of the INK4 family and the Cip/Kip family, each of which comprise three to four members, respectively (Sherr, C. J. and J. M. Roberts., 1995, Genes Dev. 9: 1149-1163). The expression of individual CDKIs is regulated independently, suggesting that each CDKI plays a unique role in the control of the cell cycle. p16INK4a, belonging to the INK4 family, binds to cyclin D and prevents the formation of catalytically active kinase complexes with the cyclin-dependent kinase (CDK) 4 or CDK6. Accordingly, p16INK4a inhibits the cell cycle at the G1/S transition (Lukas, J. et al., 1995, Nature 375: 503-506; Koh, J. et al., 1995, Nature 375: 506-510; Serrano, M., 1997, Exp. Cell Res. 237: 7-13). p21Cip1, which belongs to the Cip/Kip family, inhibits a wide variety of cyclin/CDK complexes (Harper, J. W. et al., 1993, Cell 75: 805-816; Xiong, Y. et al., 1993, Nature 366: 701-704). p21Cip1 also binds to the proliferating cell nuclear antigen (PCNA), which activates DNA polymerase δ and inactivates PCNA (Flores-Rozas, H. et al., 1994, Proc. Natl. Acad. Sci. USA. 91: 8655-8659; Nakanishi, M. et al., 1995, J. Biol. Chem. 270: 17060-17063; Waga, S. et al., 1994, Nature 369: 574-578; Li, R. et al., 1994, Nature 371: 534-537). As described above, p21Cip1 inactivates the kinase activity of cyclin/CDK complexes at various stages of cell cycle and simultaneously inhibits DNA replication.
The present inventors have previously studied the expression of CDKIs in rheumatoid synovial tissues (Taniguchi, K. et al., 1999, Nature Med. 5: 760-767). CDKI belong to a group of intranuclear molecules. The inventors found that rheumatoid synovial fibroblasts (RSFs) derived from rheumatoid synovial tissues do not express CDKI p16INK4a in vivo, but readily express them when their growth is inhibited in vitro. Specific induction of p16INK4a in RSFs derived from RA patients was observed. Therefore, the inventors conducted experiments wherein the p16INK4a gene was transfected by means of an adenovirus into the joints of rats with adjuvant arthritis (AA). This administration successfully suppressed the synovial hyperplasia and other associated pathology of arthritis (Taniguchi, K. et al., 1999, Nature Med. 5: 760-767). The arthritis treatment by p16INK4a induction compares well with conventional anti-rheumatic drugs and recently developed biological reagents.
On the other hand, p21Cip1 is a CDKI belonging to a different family than that of p16INK4a. p21CiP1, as well as p16INK4a, are not expressed in vivo in fibroblasts derived from synovial tissue of rheumatism, but their expression is induced in vitro when the growth of fibroblasts is inhibited. However, unlike p16INK4a, the induction of p21Cip1 is also observed in fibroblasts of nonrheumatoid origin (Taniguchi, K. et al., 1999, Nature Med. 5: 760-767). Additionally, in comparison to p16INK4a, p21Cip1 inhibits all kinds of CDKs. Forced expression of p21Cip1 also arrests cell cycle of normal and tumor cells at the G1 phase (Dimri, G. P. et al., 1996, Mol. Cell. Biol. 16: 2987-2997). The p16INK4a protein level gradually rises and stays high in the senescent cells. In contrast, the expression level of p21Cip1 in fibroblasts goes up with an increase in cell division, and decreases when the cells approach senescence (Tahara, H. et al., 1995, Oncogene 10: 835-840). Although p16INK4a gene expression appears to have a more direct impact in senescence induction (Alcorta, D. A. et al., 1996, Proc. Natl. Acad. Sci. USA 93: 13742-13747), the biological function of p21Cip1 is more complex. In certain settings, p21Cip1 promotes the formation of active kinase complexes by cyclin and CDK, and promotes the cell cycle rather than stopping it (LaBaer, J. et al., 1997, Genes Dev. 11: 847-862). Whereas the N-terminal domain of p21Cip1 interacts with CDK/cyclin complexes, the C-terminal domain binds to and inhibits the proliferation cell nuclear antigen (PCNA), a subunit of DNA polymerase δ essential for DNA replication and repair (Li, R. et al., 1994, Nature 371: 534-537). Furthermore, the expression of p21Cip1 enhances NF-κB-dependent gene expression by inhibiting cyclin E/CDK2, which binds to the complexes of p300/CBP coactivators and NF-κB (Perkins, N. D. et al., 1997, Science 275: 523-527). In addition, the forced expression of p21Cip1 in certain cell lines was shown to induce apoptotic cell death (Tsao, Y. P. et al., 1999, J. Virol. 73: 4983-4990; Matsushita, H. et al., 1998, Hypertension 31: 493-498; Kondo, Y. et al., 1997, Exp. Cell Res. 236: 51-56; Sheikh, M. S. et al., 1995, Oncogene 11: 1899-1905). Moreover, biological effects, other than inhibition of kinase activity, are distinct between p16INK4a and p21Cip1 (LaBaer, J. et al., 1997, Genes Dev. 11: 847-862; Li, R. et al., 1994, Nature 371: 534-537; Xiong, Y. et al., 1993, Nature 366: 701-704). Disruption of the p16INK4a gene, which results in frequent tumor development in a murine model, is also different from the result obtained by p21Cip1 gene disruption (Serrano, M. et al., 1996, Cell 85: 27-37). Thus, it was not known if p21Cip1, which is distinguished structurally from p16INK4a and also differs in its mode of expression and inhibition of cell cycle, had a therapeutic effect on rheumatoid arthritis.